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Tropism-Modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

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Tropism-Modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy Vector engineering and delivery © The American Society of Gene & Cell Therapy original article original article Capsid-modified AAV for Cutaneous Gene Transfer © The American Society of Gene & Cell Therapy Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy Jessica Sallach1,2, Giovanni Di Pasquale3, Fernando Larcher4, Nadine Niehoff1,5, Matthias Rübsam1,5, Anke Huber1,2, Jay Chiorini3, David Almarza4,10, Sabine A Eming1,5,6, Hikmet Ulus7, Stephen Nishimura8, Ulrich T Hacker9, Michael Hallek1,2, Carien M Niessen1,5,6 and Hildegard Büning1,2 1Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; 2Department I for Internal Medicine, University Hospital Cologne, Cologne, Germany; 3Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Insti- tutes of Health, Bethesda, Maryland, USA; 4Centro de Investigacion Energetica Medioambiental y Tecnologica (CIEMAT) Madrid, Center for Biomedical Research on Rare Diseases (CIBERER-U714) and Department of Bioengineering. Universidad Carlos III de Madrid (UC3M), Madrid, Spain; 5Depart- ment of Dermatology, University Hospital of Cologne, Cologne, Germany; 6Cologne Excellence Center on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany; 7Children’s Medical Surgery, Cologne, Germany; 8Department of Pathology & Laboratory Medicine, Anatomic Pathology, UCSF, San Francisco, CA, USA; 9University Cancer Center Leipzig (UCCL), Leipzig, Germany; 10Current address: Molecular Immunology, UCL Institute of Child Health, London, UK. Autologous human keratinocytes (HK) forming sheet and a strictly regulated spatiotemporal terminal differentiation grafts are approved as skin substitutes. Genetic engineer- program, which ultimately leads to the formation of the stratum ing of HK represents a promising technique to improve corneum, representing a water impermeable layer of avital corni- engraftment and survival of transplants. Although effica- fied cells.3 cious in keratinocyte-directed gene transfer, retro-/lenti- Insults to the barrier including skin wounding initiate a viral vectors may raise safety concerns when applied in well-coordinated process to restore skin barrier function that— regenerative medicine. We therefore optimized adeno- depending on the insult—includes cell proliferation, migration, associated viral (AAV) vectors of the serotype 2, charac- extracellular matrix deposition, angiogenesis, and skin remod- terized by an excellent safety profile, but lacking natural eling.4 This process fails in conditions of large cutaneous burns tropism for HK, through capsid engineering. Peptides, and chronic wounds. Limiting factors are the size of damage, an selected by AAV peptide display, engaged novel recep- altered, inflammatory environment and/or lack of proper angio- tors that increased cell entry efficiency by up to 2,500- genesis. Regenerative medicine has shown therapeutic promise in fold. The novel targeting vectors transduced HK with these settings. Specifically, autologous keratinocytes are grown on high efficiency and a remarkable specificity even in mixed either artificial or natural supporting scaffolds and skin sheets are cultures of HK and feeder cells. Moreover, differentiated subsequently transplanted. Engraftment as well as wound closure keratinocytes in organotypic airlifted three-dimensional can be significantly improved if the transplant secretes antiin- cultures were transduced following topical vector appli- flammatory cytokines and/or growth factors including angiogenic 5 cation. By exploiting comparative gene analysis we fur- mediators. These factors are only transiently required arguing for ther succeeded in identifying αvβ8 integrin as a target the use of nonintegrating vectors for gene delivery. receptor thus solving a major challenge of directed evo- Adeno-associated virus (AAV)-based vectors—classified as lution approaches and describing a promising candidate nonintegrating vectors—have emerged as one of the most prom- 6 3September2013 receptor for cutaneous gene therapy. ising delivery system for clinical use. They are of low immuno- genicity, can be produced at very high titers and deliver DNA Received 3 September 2013; accepted 11 January 2014; advance online vector genomes, which form episomes serving as templates for 11January2014 publication 25 February 2014. doi:10.1038/mt.2014.14 transcription.7 The prototype AAV vector is based on the serotype 10.1038/mt.2014.14 INTRODUCTION 2 (AAV2). It can be pseudotyped with capsids of other natural The skin is a highly specialized barrier that protects the organ- occurring serotypes or with engineered capsids.7 Molecular Therapy ism from environmental challenges while preventing loss of water AAV vectors have been successfully applied in over 80 clini- and small nutrients.1,2 The skin’s most outer layer, the epidermis, cal trials with an excellent safety profile.7 Clinical benefit was 22 is a stratified, multilayered epithelium consisting of the stratum reported for the treatment of a range of inherited diseases such as basale, the stratum spinosum, the stratum granulosum and the hemophilia B, Leber’s congenital amaurosis or lipoprotein lipase stratum corneum.1,2 To maintain proper protection against dehy- deficiency.6 The use of this vector system for the treatment of 5 dration and the environment, the barrier is not static but requires inherited skin diseases or in impaired wound healing, however, continuous renewal. This is achieved by a tightly controlled bal- has been hindered by the refractoriness of primary human kerati- 929 ance between lifelong self-renewal of cells in the stratum basale nocytes (HK) towards AAV-mediated transduction.8–12 939 The first two authors contributed equally to this work. Correspondence: Hildegard Büning, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 21, 50931 Cologne, 00may2014 Germany. E-mail: [email protected] 25February2014 Molecular Therapy vol. 22 no. 5, 929–939 may 2014 929 © The American Society of Gene & Cell Therapy Capsid-modified AAV for Cutaneous Gene Transfer Poor transduction activity can be addressed by directing a 100 vectors towards a novel receptor that is expressed on the target cell, mediates vector internalization and induces a cellular pro- 90 gram leading to nuclear delivery of vector genomes and transgene 80 expression.7 To this end, vectors can be equipped with ligands that 70 are either noncovalently linked or genetically incorporated into 60 50 viral capsid or envelope proteins, a technology termed cell entry xpressing cells targeting or transductional targeting. 40 Also, the capsid of AAV2 tolerates genetic incorporation of 30 peptide ligands.7 It is a 60mer composed of three different viral % of GFP-e 20 proteins (VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa)), which 10 share most of their amino acid sequence.13 This common region 0 (“common VP3 region”) is frequently exploited for insertion of Donor 1 Donor 2 Donor 3 peptide ligands as it forms the capsid exterior.14 Of particular b interest is amino acid position 587 located at the tip of a peak at the 100 ανβ5 15 capsid’s threefold symmetry axis. Its exposed position facilitates 90 α5β1 receptor binding of inserted peptides. Furthermore, insertions at 80 HSPG this site disrupt the primary receptor-binding motif, a prerequisite 70 16–18 for approaches aiming to redirect the viral tropism. 60 e cells Rational design-based approaches to modify the viral tro- 50 pism are challenged by the limited knowledge on ligands capable 40 of directing target cell transduction. We and others have therefore % of positiv 30 developed a high-throughput screening technique, the AAV peptide 20 display, to select in a directed evolution approach and within the physiological context of the viral capsid for appropriate ligands.19–21 10 Here, we have exploited this technology to develop AAV tar- 0 HK HeLa geting vectors with a striking tropism for primary HK. In contrast to natural occurring AAVs, these novel vectors efficiently trans- Figure 1 Lack of receptor expression renders primary human kera- duce HK in monocultures, show a remarkable specificity for HK tinocytes (HK) refractory to transduction by AAV2. (a) Primary HK in feeder layer cocultures and can be applied topically on airlifted isolated from foreskins of three different healthy donors were incubated with 5 × 103 vector genome containing particles (vg) per cell of AAV2. three-dimensional (3D) organotypic skin cultures for transduc- The AAV2 vector encoded for enhanced green fluorescent protein (GFP) tion of differentiated keratinocytes. The latter feature is not only in a self-complementary vector genome conformation. Percentage of important in basic research, but represents a central prerequisite transgene expressing cells was quantified by flow cytometry 48 hours posttransduction. Mean of three independent experiments ± SD is for future clinical applications. Finally, we established compara- shown. (b) HK and, as control, the cervix carcinoma cell line HeLa were tive gene analysis as a tool to identify receptors targeted by vectors stained with mouse antihuman αvβ5, mouse antihuman α5β1 or mouse selected in high-throughput screens. We thereby solved a major antihuman heparan sulfate proteoglycan (HSPG) antibody, followed by problem impeding the application of tropism-modified vectors a phycoerythrin-labeled IgG secondary antibody. The percentage of cells positive for HSPG
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